Difference between revisions of "Team:Rice/Wet Lab"

Line 1: Line 1:
−
 
−
 
 
{{Rice}}
 
{{Rice}}
 
<html>
 
<html>
−
 
 
<head>
 
<head>
 
<style>
 
<style>
Line 71: Line 68:
 
<script src="https://ajax.googleapis.com/ajax/libs/jquery/1.12.4/jquery.min.js"></script>
 
<script src="https://ajax.googleapis.com/ajax/libs/jquery/1.12.4/jquery.min.js"></script>
 
</head>
 
</head>
−
 
 
<body>
 
<body>
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
    <br><br><br><br><br><br><br><br><br><br><br><br><br><br>
−
  <div class="fixed_flyer" id = "sec1" style="position:relative;z-index:1">
+
    <div class = "fixed_flyer" id = "sec1" style="position:relative;z-index:8;">
−
    <div class = "h1"><a >Overview</a></div>
+
        <div class = "h1" style="color:white">Introduction</div>
−
  </div>
+
    </div>
−
  <div class="pagediv">
+
   
−
  <br>
+
    <div class="pagediv">
−
  <div class="para">
+
      <br><br>
−
  Photoacoustic imaging is a technique in which contrast agents absorb photon energy and emit signals that can be analyzed by ultrasound. Currently, photoacoustics is used to image blood vessels because heme is a natural contrast agent found in blood. Photoacoustic imaging also provides a non-invasive alternative to current diagnostic tools used to detect internal tissue inflammation. In previous literature, hypoxia and nitric oxide have both been discovered to molecularly indicate gut inflammation, and iRFP670, 713, anacy and cyan have been found to emit wavelengths that are different from heme and can penetrate tissue with near-infrared wavelengths. Therefore, our goal is to report inflammation and cancer in the gut through photoacoustic imaging of engineered E. coli that express bacterial pigment violacein, as well as near-infrared fluorescent proteins iRFP670 and iRFP713.
+
      <div class = "para">We facilitated a Building with Biology event at the Children’s Museum of Houston. Through hands-on activities like extracting DNA from wheat germ and helping kids to design their own superorganisms, we had the opportunity to introduce young children to the exciting field of synthetic biology and to clear up a few of their parents’ misconceptions.
−
 
+
−
<br><br>
+
−
  </div>
+
−
  </div>
+
−
 
+
−
 
+
−
  <div class="fixed_flyer" id = "sec2" style="position:relative;z-index:2">
+
−
    <div class = "h1">Arabinose Induced iRFP 670 and 713 Fluorescence</div>
+
−
  </div>
+
−
  <div class="pagediv">
+
−
  <br>
+
−
<div class="para">
+
−
pBAD is a very well-characterized expression system in E. coli. pBAD normally works by arabinose induction: araC, a constitutively produced transcription regulator, changes form in the presence of arabinose sugar, allowing for the activation of promoter pBAD. Therefore, we formed genetic circuits consisting of the pBAD expression system and iRFP670 and 713 to test the inducibility of our iRFPs. <br><br>
+
−
  </div>
+
−
  </div>
+
−
 
+
−
 
+
−
  <div class="fixed_flyer" id = "sec3" style="position:relative;z-index:3">
+
−
    <div class = "h1">Nitric-oxide-induced Fluorescence</div>
+
−
  </div>
+
−
  <div class="pagediv">
+
−
    <br>
+
−
<div class="para">
+
−
The next step was to test the nitric oxide induction of iRFP fluorescence. We used a genetic circuit consisting of a constitutive promoter that always expresses Part:BBa_K554003, which encodes for the expression of a SoxR. In the presence of nitric oxide, SoxR changes form to activate the promoter SoxS, which in turn is supposed to activate the expression of the iRFPs. Thus, for the next assay we added DETA/NO, a nitric oxide adduct in the presence of water.  
+
−
 
+
 
</div>
 
</div>
−
</div>
+
      <br><br>
−
 
+
    </div>
−
<br><br>
+
    <div class = "fixed_flyer" id = "sec2" style="position:relative;z-index:9;">
−
  <div class="fixed_flyer" id = "sec4" style="position:relative;z-index:4">
+
        <div class = "h1" style="color:white">Background</div>
−
    <div class = "h1">Hypoxia induced fluorescence</div>
+
    </div>
−
  </div>
+
    <div class="pagediv" style="
−
  <div class="pagediv">
+
    height: 700px;
 +
">
 +
      <br><br>
 +
      <div class = "para" style="text-align:left;width: 565px;">The Building with Biology project  is dedicated to spreading STEM learning and discussion about the technological and societal implications of synthetic biology through public and scientist dialogue. Successful applicants receive a physical kit with all of the supplies necessary to host an event with six hands-on activities.
 +
      </div>
 +
      <ol style = "text-align: left; width:530px">
 +
        <li><div class = "para">Bio Bistro: Decide what current and future synthetic biology-based food products you would, would not, or might eat.</div></li>
 +
        <li><div class = "para">Kit of Parts: Solve challenges by building a model cell with standardized genetic parts (like BioBricks).</div></li>
 +
        <li><div class = "para">See DNA: Extract visible DNA from wheat germ and create necklaces to display your own sample of wheat germ DNA.</div></li>
 +
        <li><div class = "para">Super Organisms: Design a superhero to rescue a person falling from a tall building and then use that same creative engineering process to design a single-celled organism to clean up an oil spill.</div></li>
 +
        <li><div class = "para">Tech Tokens: Consider the potential advantages and disadvantages of various areas of synthetic biology research, before investing in them with "tech tokens."</div></li>
 +
        <li><div class = "para">VirEx Delivery: Explore the potential for engineered viruses to deliver beneficial, targeted genetic information to sites throughout the body.</div></li>
 +
      </ol>
 +
<img src="https://static.igem.org/mediawiki/2016/0/01/Human_Practices1.jpg" width="470px" style="position: relative;top: -485px;left: 410px;">
 +
    </div>
 +
      <br><br>
 +
        <div class = "fixed_flyer" id = "sec3" style="position:relative;z-index:10;">
 +
        <div class = "h1" style="color:white">Timeline</div>
 +
    </div>
 +
    <div class="pagediv">
 +
      <br><br>
 +
<div class = "para" style="float:left">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</div>
 +
    <div id ="one"style="height:20px; width:50px;background:#551A8B;display:block;float:left"></div>
 +
<div id ="two"style="height:20px; width:200px;background:#8A2BE2;display:block;float:left"></div>
 +
<div id="three"style="height:20px; width:50px;background:#9370DB;display:block;float:left"></div>
 +
<div id ="four"style="height:20px; width:50px;background:#551A8B;display:block;float:left"></div>
 +
<div id ="five"style="height:20px; width:40px;background:#8A2BE2;display:block;float:left"></div>
 +
<div id="six"style="height:20px; width:60px;background:#9370DB;display:block;float:left"></div>
 +
<div id="seven"style="height:20px; width:20px;background:#551A8B;display:block;float:left"></div>
 
<br>
 
<br>
−
<div class="para">
+
<div id = "info">
−
 
+
  <div class = "para" id = "defaulti" style="display:box">Above is a timeline for Building with Biology, hover over different sections to learn more!</div>
−
<br>
+
  <div class = "para" id = "onei" style="display:none">Volunteer orientation at the Children’s Museum & Building with Biology training</div>
−
In addition to the sinduction of iRFP fluorescence by nitric oxide, we also tested the induction of iRFP fluorescence with a hypoxia promoter. We expected iRFP fluorescence to increase with increased hypoxic conditions (less oxygen) when using NarK promoter and fdhf promoters, both characterized as hypoxia-inducible.
+
  <div class ="para" id = "twoi"style="display:none">Studying the background materials for our respective stations and practicing our presentations.</div>
−
 
+
  <div class = "para" id = "threei"style="display:none">Set up</div>
−
Transcription of the fdhf promoter is regulated by an RNA polymerase with sigma factor 54 whose binding is dictated by presence of an additional activator complex consisting of FhlA and formate. Only when the FhlA-formate complex is present will the sigma-54 polymerase initiate transcription. This process is induced by formate, but is also heavily repressed by presence of oxygen, giving it characterization as a hypoxia sensor.  
+
  <div class = "para" id = "fouri"style="display:none">Building with Biology Event</div>
−
 
+
  <div class = "para" id = "fivei"style="display:none">Clean up</div>
−
 
+
  <div class = "para" id = "sixi"style="display:none">Debrief</div>
−
 
+
  <div class = "para" id = "seveni"style="display:none">Building with Biology Facilitator Feedback Survey</div> 
−
 
+
 
</div>
 
</div>
−
 
−
</div>
 
−
  <div class="fixed_flyer" id = "sec6" style="position:relative;z-index:6">
 
−
    <div class = "h1">Discussion</div>
 
−
  </div>
 
−
 
−
  <div class="pagediv">
 
−
  <br>
 
−
  <div class="para">
 
−
The current model is not able to show the expected dependence of violacein yield on promoter strength. After reevaluating our assumptions, we identified some potential flaws of the model that might cause the unexpected results.
 
−
<br><br>
 
−
One of the assumptions from our model is that the rate of production of L-tryptophan is constant and independent of the promoter strength. Jones el al. suggest that the L-tryptophan production rate may be affected by the metabolic burden of the production of the recombinant enzymes (VioA, VioB, etc.). This phenomenon may be caused by the depletion of essential metabolic resource, such as amino acids, mRNA and ATP. Therefore, the L-tryptophan production rate might need to be dependent on enzymes production rates.
 
−
<br><br>
 
−
Another effect that we didn’t consider is the saturation of the enzymes. To improve our model, we could include these effects by employing Michaelis-Menten Kinetics equations in our next step. Nevertheless, we have been cautious about including this in our model, since increasing the number of parameters, without increasing the number of data points usually causes the overfitting of the model.
 
−
<br><br>
 
−
Finally, since the violacein pathway has not been fully characterized, it is possible that we ignored some reactions in the complete pathway. Moreover, there may be feedback loops that regulate the pathway. We will need to investigate these possible components and incorporate them into our model if they prove to be present in the pathway.
 
−
<br><br>
 
−
  </div>
 
 
     </div>
 
     </div>
−
 
+
      <br><br>
−
     <div class="fixed_flyer" id = "sec7" style="position:relative;z-index:7">
+
     <div class = "fixed_flyer" id = "sec4" style="position:relative;z-index:11;">
−
      <div class = "h1">Conclusion</div>
+
        <div class = "h1" style="color:white">Significance</div>
 
     </div>
 
     </div>
 
     <div class="pagediv">
 
     <div class="pagediv">
−
    <br>
+
      <br><br>
−
     <div class="para">
+
     <div class = "para">The afternoon we spent at the Children’s Museum was particularly rewarding because we had the opportunity to interact with a cross section of the general public. The attendees had not come to the museum expecting to learn about synthetic biology, but they were eager to actively participate in the activities and to engage in conversations about the potential impact of the field. We were reminded by their vehement objections to buzzwords like “GMOs” of the significant resistance that synthetic biology still faces. </div>
−
    Here we present a method to fit a model of violacein production in E.coli to experimental data of violacein yield with different promoters using nonlinear regression. Although  it fails to calculate the dependence on promoter strength, our model is able predict the average violacein concentration. We expect that small changes on the model, such as including a L-tryptophan production dependence of the metabolic burden, would allow us to successfully predict the violacein production in response to the variation of promoter strength. Once the predictive model is complete, we will be able to find the strains that lead to optimal violacein yield computationally.
+
−
    </div>
+
 
     </div>
 
     </div>
 +
</body>
  
−
  <div class="fixed_flyer" id = "sec8" style="position:relative;z-index:8">
 
−
    <div class = "h1">References</div>
 
−
  </div>
 
−
  <div class="para">
 
−
 
−
<ol>
 
−
<li>Carvalho, D. D., Costa, F. T. M., Duran, N., & Haun, M. (2006). Cytotoxic activity of violacein in human colon cancer cells. <i>Toxicology in Vitro</i>, 20(8), 1514–1521. <br><a href="http://dx.doi.org/10.1016/j.tiv.2006.06.007">http://dx.doi.org/10.1016/j.tiv.2006.06.007</a></li>
 
−
<li>Jones, J. A., Vernacchio, V. R., Lachance, D. M., Lebovich, M., Fu, L., Shirke, A. N., … Koffas, M. A. G. (2015). ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. <i>Scientific Reports</i>, 5, 11301. <br><a href="http://doi.org/10.1038/srep11301">http://doi.org/10.1038/srep11301</a></li>
 
−
<li>Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J., & Dueber, J. E. (2013). Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay. <i>Nucleic Acids Research</i>, 41(22), 10668–10678. <br> <a href="http://doi.org/10.1093/nar/gkt809">http://doi.org/10.1093/nar/gkt809</a></li>
 
−
</ol>
 
−
</div>
 
−
</div>
 
−
<br><br><br><br><br>
 
−
</body>
 
 
<script>
 
<script>
 
$(document).ready(function(){
 
$(document).ready(function(){
Line 180: Line 145:
 
     var sec4=$(".fixed_flyer#sec4");
 
     var sec4=$(".fixed_flyer#sec4");
 
     var sec4_pos=sec4.offset().top;
 
     var sec4_pos=sec4.offset().top;
−
    var sec5=$(".fixed_flyer#sec5");
 
−
    var sec5_pos=sec5.offset().top;
 
−
    var sec6=$(".fixed_flyer#sec6");
 
−
    var sec6_pos=sec6.offset().top;
 
−
    var sec7=$(".fixed_flyer#sec7");
 
−
    var sec7_pos=sec7.offset().top;
 
−
    var sec8=$(".fixed_flyer#sec8");
 
−
    var sec8_pos=sec8.offset().top;
 
 
     $(window).scroll(function () {
 
     $(window).scroll(function () {
 
         var y=$(this).scrollTop();
 
         var y=$(this).scrollTop();
Line 196: Line 153:
 
         console.log('sec3:', sec3_pos);
 
         console.log('sec3:', sec3_pos);
 
         console.log('sec4:', sec3_pos);
 
         console.log('sec4:', sec3_pos);
−
         if(y<sec2_pos-50){
+
         if(y<sec2_pos-40){
 
           if (y>sec1_pos){
 
           if (y>sec1_pos){
 
             console.log('sec 1 supposed to move');
 
             console.log('sec 1 supposed to move');
−
             sec1.stop().animate({'top':y-sec1_pos+18},1);
+
             sec1.stop().animate({'top':y-sec1_pos},1);  
 
           }
 
           }
 
         } else {
 
         } else {
−
           sec1.stop().animate({'top':10},1);
+
           sec1.stop().animate({'top':10},1);    
 
         };
 
         };
−
         if(y<sec3_pos-50){
+
         if(y<sec3_pos-40){
 
           if(y>sec2_pos){
 
           if(y>sec2_pos){
 
             console.log('sec 2 supposed to move');
 
             console.log('sec 2 supposed to move');
−
             sec2.stop().animate({'top':y-sec2_pos+18},1);
+
             sec2.stop().animate({'top':y-sec2_pos},1);
 
           }
 
           }
 
         } else {
 
         } else {
−
           sec2.stop().animate({'top':10},1);
+
           sec2.stop().animate({'top':10},1);    
 
         };
 
         };
 
         if(y<sec4_pos-40){
 
         if(y<sec4_pos-40){
 
           if(y>sec3_pos){
 
           if(y>sec3_pos){
 
             console.log('sec 3 supposed to move');
 
             console.log('sec 3 supposed to move');
−
             sec3.stop().animate({'top':y-sec3_pos+18},1);
+
             sec3.stop().animate({'top':y-sec3_pos},1);
 
           }
 
           }
 
         } else {
 
         } else {
−
           sec3.stop().animate({'top':10},1);
+
           sec3.stop().animate({'top':10},1);    
 
         };
 
         };
−
         if(y<sec5_pos-40){
+
         if(y<3000){
 
           if(y>sec4_pos){
 
           if(y>sec4_pos){
 
             console.log('sec 4 supposed to move');
 
             console.log('sec 4 supposed to move');
−
             sec4.stop().animate({'top':y-sec4_pos+18},1);
+
             sec4.stop().animate({'top':y-sec4_pos},1);
 
           }
 
           }
 
         } else {
 
         } else {
−
           sec4.stop().animate({'top':10},1);
+
           sec4.stop().animate({'top':10},1);    
−
        };
+
−
        if(y<sec6_pos-40){
+
−
          if(y>sec5_pos){
+
−
            console.log('sec 5 supposed to move');
+
−
            sec5.stop().animate({'top':y-sec5_pos+18},1);
+
−
          }
+
−
        } else {
+
−
          sec5.stop().animate({'top':10},1);
+
−
        };
+
−
        if(y<sec7_pos-40){
+
−
          if(y>sec6_pos){
+
−
            console.log('sec 6 supposed to move');
+
−
            sec6.stop().animate({'top':y-sec6_pos+18},1);
+
−
          }
+
−
        } else {
+
−
          sec6.stop().animate({'top':10},1);
+
−
        };
+
−
        if(y<sec8_pos-40){
+
−
          if(y>sec7_pos){
+
−
            console.log('sec 7 supposed to move');
+
−
            sec7.stop().animate({'top':y-sec7_pos+18},1);
+
−
          }
+
−
        } else {
+
−
          sec7.stop().animate({'top':10},1);
+
−
        };
+
−
        if(y<5000){
+
−
          if(y>sec8_pos){
+
−
            console.log('sec 8 supposed to move');
+
−
            sec8.stop().animate({'top':y-sec8_pos+18},1);
+
−
          }
+
−
        } else {
+
−
          sec8.stop().animate({'top':10},1);
+
 
         };
 
         };
 +
    });
 +
        $("#one").hover(function(){
 +
        $("#onei").css("display", "block");
 +
        $("#one").css("opacity", "0.5");
 +
        $("#defaulti").css("display","none");
 +
        }, function(){
 +
        $("#onei").css("display","none");
 +
        $("#one").css("opacity", "1");
 +
        $("#defaulti").css("display","block");
 +
    });
 +
        $("#two").hover(function(){
 +
        $("#twoi").css("display", "block");
 +
        $("#two").css("opacity", "0.5");
 +
        $("#defaulti").css("display","none");
 +
        }, function(){
 +
        $("#twoi").css("display","none");
 +
        $("#two").css("opacity", "1");
 +
        $("#defaulti").css("display","block");
 +
    });
 +
        $("#three").hover(function(){
 +
        $("#threei").css("display", "block");
 +
        $("#three").css("opacity", "0.5");
 +
        $("#defaulti").css("display","none");
 +
        }, function(){
 +
        $("#threei").css("display","none");
 +
        $("#three").css("opacity", "1");
 +
        $("#defaulti").css("display","block");
 +
    });
 +
        $("#four").hover(function(){
 +
        $("#fouri").css("display", "block");
 +
        $("#four").css("opacity", "0.5");
 +
        $("#defaulti").css("display","none");
 +
        }, function(){
 +
        $("#fouri").css("display","none");
 +
        $("#four").css("opacity", "1");
 +
        $("#defaulti").css("display","block");
 +
    });
 +
        $("#five").hover(function(){
 +
        $("#fivei").css("display", "block");
 +
        $("#five").css("opacity", "0.5");
 +
        $("#defaulti").css("display","none");
 +
        }, function(){
 +
        $("#fivei").css("display","none");
 +
        $("#five").css("opacity", "1");
 +
        $("#defaulti").css("display","block");
 +
    });
 +
        $("#six").hover(function(){
 +
        $("#sixi").css("display", "block");
 +
        $("#six").css("opacity", "0.5");
 +
        $("#defaulti").css("display","none");
 +
        }, function(){
 +
        $("#sixi").css("display","none");
 +
        $("#six").css("opacity", "1");
 +
        $("#defaulti").css("display","block");
 +
    });
 +
        $("#seven").hover(function(){
 +
        $("#seveni").css("display", "block");
 +
        $("#seven").css("opacity", "0.5");
 +
        $("#defaulti").css("display","none");
 +
        }, function(){
 +
        $("#seveni").css("display","none");
 +
        $("#seven").css("opacity", "1");
 +
        $("#defaulti").css("display","block");
 
     });
 
     });
 
   });
 
   });
 
</script>
 
</script>
−
 
 
</html>
 
</html>

Revision as of 02:46, 22 November 2016















Introduction


We facilitated a Building with Biology event at the Children’s Museum of Houston. Through hands-on activities like extracting DNA from wheat germ and helping kids to design their own superorganisms, we had the opportunity to introduce young children to the exciting field of synthetic biology and to clear up a few of their parents’ misconceptions.


Background


The Building with Biology project is dedicated to spreading STEM learning and discussion about the technological and societal implications of synthetic biology through public and scientist dialogue. Successful applicants receive a physical kit with all of the supplies necessary to host an event with six hands-on activities.
  1. Bio Bistro: Decide what current and future synthetic biology-based food products you would, would not, or might eat.
  2. Kit of Parts: Solve challenges by building a model cell with standardized genetic parts (like BioBricks).
  3. See DNA: Extract visible DNA from wheat germ and create necklaces to display your own sample of wheat germ DNA.
  4. Super Organisms: Design a superhero to rescue a person falling from a tall building and then use that same creative engineering process to design a single-celled organism to clean up an oil spill.
  5. Tech Tokens: Consider the potential advantages and disadvantages of various areas of synthetic biology research, before investing in them with "tech tokens."
  6. VirEx Delivery: Explore the potential for engineered viruses to deliver beneficial, targeted genetic information to sites throughout the body.


Timeline


                               

Above is a timeline for Building with Biology, hover over different sections to learn more!


Significance


The afternoon we spent at the Children’s Museum was particularly rewarding because we had the opportunity to interact with a cross section of the general public. The attendees had not come to the museum expecting to learn about synthetic biology, but they were eager to actively participate in the activities and to engage in conversations about the potential impact of the field. We were reminded by their vehement objections to buzzwords like “GMOs” of the significant resistance that synthetic biology still faces.